The disclosed invention is in the general field of nucleic acid detection, and specifically in the field of detection of methylation of nucleic acids using quantitative analysis.
Successful implementation of the promise of personalized medicine lies at the crossroads of performing accurate, rapid genetic analyses with the interpretation of the test results to diagnose and treat individual patients. The critically important parameter among these qualities is the accuracy of diagnostic test results.
DNA methylation is a ubiquitous mechanism of epigenetic regulation. Under normal physiological conditions, methylation is involved in many functions, including development, suppressing parasitic sequences, silencing incidental promoters, propagating epigenetic inheritance, and marking the inactive X chromosome. In particular, the promoters of many crucial genes contain CG-rich regions called CpG islands. These islands are usually nonmethylated; methylation of these regions results in silencing of the gene's expression. Cancerous (transformed) cells often show hypermethylation of these regions. In transformed cells, methylation of the O6-methylguaninemethyltrasferase (MGMT) enzyme promoter is associated with favorable response to alkylating chemotherapies. These alkylating chemotherapies are often used, along with radiation, in battling glioblastoma multiforme (GBM), a common brain cancer with a dismal (0-5%) survival rate. Methylated cytidines (m5CpG) are therefore important biomarkers for transposable elements, viral DNA, intra-ORF promoter sequences, silenced genes, cancerous tissue and cancer treatment prognoses.
Current methods of evaluating DNA methylation include restriction digests, bisulfite treatment followed by QPCR, sequencing or microarray, and immunoprecipitation or affinity chromatography followed by microarray. The first two approaches are labor intense and technically complicated, and bisulfite treatment is time-consuming. The remaining methods are state-of-the-art, but provide less specific information and are only semi-quantitative. Therefore, in both research and clinical applications, there is need for a rapid, quantitative, and highly specific assay to measure methylation levels while retaining the massive parallelism of microarray technology.
Microarray-formatted DNA methylation assays already exist, but they are inadequate for several reasons. The standard methodology for evaluating microarray hybridization reactions entails incubating a patient's sample for nominally 18 hours and analyzing the DNA-DNA binding results with a single endpoint measurement. This measurement is taken at what is assumed to be the hybridization reaction's equilibrium point. Indeed, there are many thousands of hybridization reactions simultaneously occurring on the microarray—each with its own and different equilibration times, thereby inherently compromising the value of the microarray data. These current microarray-based technologies are also not quantitative, in part because of imperfect molecular recognition (cross-hybridization). This drawback limits their use to tasks such as qualitative (and all too often unreliable) “noisy” screening. Due to these limitations, current microarray techniques attempt to compensate through excessive redundancy. All of these factors have caused the major problem with microarray technology and the genetic problems it has been employed to elucidate—i.e., managing great quantities of inaccurate, irreproducible data. This standard is unacceptable for use in evaluating a patient's DNA for a deadly disease such GBM. Additionally, sorting through copious amounts of typical microarray data profoundly extends analysis time, results in improper conclusions, and initiates interpretive controversy.
Methyl binding domain (MBD) proteins have been shown to recognize methylated DNA both in vitro and in vivo. Recently, protein that binds to symmetrically methylated CpG sites with high affinity and specificity has been engineered by expressing only the methyl binding domain of the protein MBD1.
What is needed in the art are methods and compositions that make use of the MBD protein to analyze methylation of nucleic acids using real time, quantitative assays.